1. Field of Invention
This invention relates to Escherichia coli, and particularly to a DNA sequence of biotin operon in which a base pair of either a nucleotide sequence of the regulatory region of the biotin operon of Escherichia coli or a nucleotide sequence in the vicinity of the bioB initiation codon is mutated in comparison with that of its wild type strain. According to such a DNA sequence, a system for highly expressing biotin operon can be constructed.
2. Description of Related Art
In the production of biotin, which is a vitamin required for animals, plants, and microorganisms, in order to substitute chemical synthetic processes involving complicated steps, effective processes utilizing a fermentation process employing microorganisms improved by genetic engineering techniques, etc. have been developed (see, for example, Japanese Unexamined Patent Publication (Kokai) No. 61-149091 and EP-A-0316229).
As highly producing strains in which a microorganism is improved to enhance the productivity of a specific useful substance, in the case where there exists a feedback repression mechanism to an enzyme synthesizing system by the final product, repressor mutants and operator mutants produced by mutation can be mentioned. Also, strains where their promotor activity per se in the enzyme synthesizing system has been reinforced have been suggested. In the case of fully making use of genetic engineering technology, there are also processes where an enzyme synthesizing system is ligated to a highly expressing promotor different from its original promotor. The repressor mutants relating to highly biotin producing strains include, for example, DRK 332 strain (FERM P-8585, Japanese Unexamined Publication (Kokai) No. 155081), and an example of using a highly expressing promoter with genetic engineering technology is a strain where only bioB gene is reinforced using a P.sub.L promotor (Japanese Unexamined Publication (Kokai) No. 61-149091), etc.
Although all of the above-enumerated microorganisms having been improved for the purpose of increasing biotin productivity have attained their desired objects, there is still room for improvement, and a necessity for providing further improved strains with high biotin productivity still remains in order to improve the productivity of biotin by fermentation methods. In the biotin operon of Escherichia coli, five genes, i.e., bioA, bioB, bioF, bioC, and bioD contributing to the biotin biosynthesis are coded. The regulatory region which controls their expression locates between bioA and bioB, and bioA is coded in the left chain, while bioB, bioF, bioC, and bioD are coded in the right chain. They are subjected to transcription in the reverse direction, respectively, and the transcription in both directions is controlled by one operator. The transcription mechanism in both directions is also one reason why the alternation into a highly expressing promotor by genetic engineering technology cannot be simplified. In spite of this, it is important for improving the productivity of biotin to attain a high expression of the biotin operon. Several reports have been made for the mutation of the operator (Nature, 276, 689 (1978), Gene, 13, 89 (1981)), but there is no mention about the productivity of biotin. In some cases, the promotor activity overlapping with the operator region is significantly decreased.